WO2005094415A2 - Vecteurs recombinants et methodes pour induire une reponse immunitaire - Google Patents

Vecteurs recombinants et methodes pour induire une reponse immunitaire Download PDF

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WO2005094415A2
WO2005094415A2 PCT/US2005/003903 US2005003903W WO2005094415A2 WO 2005094415 A2 WO2005094415 A2 WO 2005094415A2 US 2005003903 W US2005003903 W US 2005003903W WO 2005094415 A2 WO2005094415 A2 WO 2005094415A2
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rna
vectors
virus
transgene
nucleic acid
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WO2005094415A3 (fr
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Scott E. Hensley
Hildegund C. J. Ertl
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The Wistar Institute
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/235—Adenoviridae
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • VA-RNA Virus-associated RNA
  • VA-RNA Virus-associated RNA
  • Ad2 VA-RNA stimulates protein synthesis in the infected cell (O'Malley, et al . (1986) Cell 44:391-400; Reichel, et al . (1985) Nature (London) 313:196-200; Schneider, et al . (1984) Cell 37:291-298; Siekierka, et al . (1985) Proc. Natl . Acad. Sci . USA 82:1959-1963) and serves to antagonize the interferon-induced cellular antiviral defense system (Kitajewski, et al .
  • Ad2 VA-RNA also stimulates the expression of transfected genes (Kaufman and
  • PKA Protein kinase R
  • This kinase has been shown to be involved during the induction of type I interferon (IF ⁇ ) in myeloid dendritic cells that are exposed to cytoplasmic double-stranded R ⁇ A (Diebold, et al . (2003) Nature 424 (6946) : 324-8) . It has been shown that VA-RNA; . molecules of various adenoviruses have the ability to bind to and inhibit PKR (Kitajewski, et al . (1986) Cell 45:195- 200; Mathews, et al . (1991) J. Virol . 65:5657-5662).
  • IF ⁇ type I interferon
  • VA-RNA molecules encoded by certain serotypes of adenovirus inhibit PKR with a much greater efficiency compared to VA-RNA molecules from other serotypes of adenovirus (Ma (1993) J. Virol . 67:6605-6617; Larsson, et al. (1986) J. Virol . 58:600-609).
  • VA-RNA; . of some serotypes of adenovirus allows for the production of high amounts of viral proteins while inhibiting the production of type I IFNs during natural infections.
  • Adenoviruses have been engineered into replication- defective vectors by deleting essential early genes.
  • a transgene can be cloned into these vectors, allowing for in vi tro or in vivo delivery of foreign genetic material.
  • Adenoviral vectors have been explored as delivery vehicles for gene therapy purposes and also vaccination approaches.
  • Most studies and clinical trials have been conducted using vectors derived from human adenovirus serotype 5 (AdHu5) or human adenovirus serotype 2 (AdHu2) .
  • AdHu5 human adenovirus serotype 5
  • AdHu2 human adenovirus serotype 2
  • One aspect of the present invention is a recombinant vector containing virus-associated RNA nucleic acid sequences.
  • the virus-associated RNA of such a vector are fusions, additions, or replacements; or insertions, deletions, point mutations, or substitutions within the virus-associated RNA nucleic acid sequences which result in an altered immune response to said vector or products encoded thereby as compared to a wild-type vector.
  • the recombinant vector contains a transgene for use in gene therapy or a vaccine.
  • Another aspect of the present invention is a method for inducing an immune response to an antigen in a subject by administering to the subject an effective amount of an antigen-encoding recombinant vector which contains virus- associated RNA nucleic acid sequences which are fusions, additions, or replacements; or contain insertions, deletions, point mutations, or substitutions within the virus-associated RNA nucleic acid sequences.
  • a further aspect of the present invention is a method for treating a genetic or acquired disease in a subject by administering to the subject an effective amount of an transgene-encoding recombinant vector which contains virus- associated RNA nucleic acid sequences which are fusions, additions, or replacements; or contain insertions, deletions, point mutations, or substitutions within the virus-associated RNA nucleic acid sequences.
  • an transgene-encoding recombinant vector which contains virus- associated RNA nucleic acid sequences which are fusions, additions, or replacements; or contain insertions, deletions, point mutations, or substitutions within the virus-associated RNA nucleic acid sequences.
  • PKR inhibitor 2-aminopurine
  • 2-aminopurine decreases the amount of type I IFN production that is stimulated after transducing splenocytes by certain serotypes of adenoviral vectors.
  • VA-RNAi transcripts are produced by adenoviral vectors, possibly via efficient transcription by RNA polymerase III.
  • VA-RNA molecules encoded by different serotypes of adenovirus have varying abilities to inhibit PKR (Ma (1993) J. Virol .
  • VA-RNA genes may result from natural variations in VA-RNA genes within each vector. Indeed, although the vectors are quite homologous, there is a disproportionate amount of sequence divergence within the VA-RNA genes.
  • AdHu5 and AdHu2 adenoviruses encoded active VA-RNA genes that were able to bind to PKR with a high affinity and inhibit its function (Ma (1993) J. Virol . 67:6605-6617).
  • AdHu5 and AdHu2 adenoviruses encoded active VA-RNA genes that were able to bind to PKR with a high affinity and inhibit its function (Ma (1993) J. Virol . 67:6605-6617).
  • AdC68 vectors may also encode an intermediate VA-RNA that does not efficiently inhibit PKR.
  • hybrid adenoviral vectors that contain VA-RNA genes from various serotypes of adenovirus may be constructed which vary in their efficiency to inhibit PKR.
  • one aspect of the invention is a recombinant vector containing VA-RNA nucleic acid sequences which are modified so that an immune response to said vector or products encoded thereby is altered as compared to a wild-type vector, wherein a wild-type vector is intended to mean the same vector which lacks a modified VARNA nucleic acid sequences or a closely related vector thereof.
  • a VA-RNA nucleic acid sequence which may be modified in the vector of the invention includes VA-RNAi and VA-RNAn; preferably VA-RNAi.
  • a recombinant vector is intended to include viral vectors as well as naked DNA vectors ⁇ e . g. , plasmids) .
  • a recombinant vector of the present invention is a viral vector.
  • a recombinant vector of the present invention may be used as a vaccine or may be used to deliver therapeutic gene products for gene therapy.
  • transgene is meant any exogenous DNA sequence which is inserted into the vector for expression in a cell into which the vector is introduced ⁇ e . g.
  • a transgene product or product of a transgene includes, but is not limited to, a protein, peptide, mRNA, ribozyme, siRNA, and the like.
  • One modification to a VA-RNA nucleic acid sequence includes the addition of one, two, three, or more heterologous or homologous VA-RNA nucleic acid sequences into a vector which contains none, one or two endogenous VA-RNA nucleic acid sequences.
  • VA-RNA nucleic acid sequences contain a promoter located within the coding sequence
  • a VA-RNA nucleic acid sequence may be inserted or cloned into a vector at any location which does not disrupt an essential function of the vector or may be inserted downstream or upstream of a selected transgene of interest ⁇ e . g. , gene encoding an antigen or gene encoding a therapeutic product) . It is contemplated that insertion of a VA-RNA nucleic acid sequence downstream or upstream of a selected transgene of interest which is regulated by a strong promoter ⁇ e. g. , a CMV promoter) will result in enhanced expression of the VA-RNA nucleic acid sequence.
  • VA-RNA nucleic acid sequences may further be modified by replacing an endogenous VA-RNA nucleic acid sequence with a homologous or heterologous VA-RNA nucleic acid sequence, preferably from another serotype ⁇ e . g. , replacing a AdHu5 VA-RNA nucleic acid sequence with an AdC68 nucleic acid sequence) .
  • a replacement VA-RNA nucleic acid sequence may be inserted or cloned into the same location as the VARNA nucleic acid sequence which is being replaced.
  • a replacement VA-RNA nucleic acid sequence may be inserted or cloned into any location of the vector ⁇ e . g.
  • VA-RNA nucleic acid sequence fusions are also considered modified VA-RNA sequences.
  • the 5' sequences of an AdHu5 VA-RNA nucleic acid sequence may be fused with the 3' sequences of an AdC68 VA-RNA nucleic acid sequence to create a fusion VA-RNA molecule. Sequences encoding such a fusion molecule may be added to a vector or replace an endogenous VA-RNA sequence. Point mutations, insertions, deletions, or substitutions are also considered modifications of VA-RNA sequences provided such modifications do not significantly impact the secondary structure of the hair-pin loop that form the RNA such that it will not fold properly.
  • VA-RNA nucleic acid sequences are well-known in the art and may be modified using standard molecular biology methods such as those described by Maniatis, et al . ((1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.) and other laboratory manuals. Exemplary VA-RNA nucleic acid sequences for use in recombinant vectors of the present invention are provided by Kidd, et al .
  • Simian adenovirus type 23 (Accession number U10684) and include, but are not limited to, Simian adenovirus type 23 (Accession number U10684) , Simian advenovirus 25 (also known as AdC68, Pan9 or SAV25; Accession number AF394196) , Human adenovirus type 5 (also referred to as AdHu5 or Ad5 ; Accession numbers BK000408, X02996 or AY339865) , Human adenovirus type 8 (Accession number U10683) , Human adenovirus type 2 (Accession number JO1917) , Human adenovirus type 4 (Accession number U10682) , Human adenovirus type 4a (Accession number U10681) , Human adenovirus type 3 (Accession number U10680) , Human adenovirus type 37 (Accession number U10679) , Human adenovirus type 35 (
  • Sequence Alignment Programs such as "Clustal W” , accessible through Web Servers on the internet.
  • Polynucleotide sequences can be compared using
  • FASTA a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences .
  • percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1. It is contemplated that the modification of VA-RNA nucleic acid sequences will have a profound impact on the immune system by, for example altering the binding efficiency to PKR and therefore the inhibition of PKR. Such recombinant vectors are therefore useful for gene therapy and for vaccine carriers. In a preferred embodiment, the recombinant vectors of the present invention are used as vaccine carriers . For example, there will be cases wherein an intermediate VA-RNA may be desirable and cases wherein an active VA-RNA will be desirable.
  • an intermediate VA-RNA is one which induces the expression of a reporter gene in the range of a three to eight-fold over background expression
  • an active VA-RNA is one which induces the expression of a reporter gene in the range of 15 to 20-fold
  • a weak VA-RNA is one which produces less than a two-fold induction in reporter gene expression.
  • vectors containing, for example, AdC68 VA-RNA nucleotide sequences may be used as AdC68 may encode an intermediate VA-RNA; .
  • VA-RNA genes removal of VA-RNA genes from adenoviral vectors is contemplated and may require the incorporation of one or more VA-RNA genes into the propagating cell line to produce such a vector.
  • vectors containing, for example, AdHu5 VA-RNA nucleotide sequences may be used as a much stronger antibody response is directed against a transgene product when vaccinating with AdHu5 vectors as compared to AdC68 vectors (Xiang, et al. (2002) J " . Virol . 76:2667-2675) . This is likely the case because strong antibody responses often require a large amount of antigen (i.e., a high level of expression) . Accordingly, VA-RNA .
  • a recombinant vector of the present invention is an adenoviral vector with preference given to subgroup C type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) , chimpanzee serotype Ad C68, Ad C6, Ad C7, or other adenoviruses of animal origin.
  • Adenoviruses of animal origin which may be used are adenoviruses of canine, bovine, murine, ovine, porcine, avian, caprine, guinea pig, fowl, fish, possum, deer or simian origin.
  • the adenovirus of animal origin is a simian or canine adenovirus .
  • Particularly suitable adenoviruses of animal origin are well-known to those of skill in the art.
  • a recombinant vector of the present invention may be either a replication-competent or a replication-defective adenovirus containing a transgene of interest ⁇ e . g. , a transgene for gene therapy or encoding an antigen of a vaccine) and further containing inverted terminal repeats and encapsidation sequence.
  • a transgene of interest ⁇ e . g. , a transgene for gene therapy or encoding an antigen of a vaccine
  • inverted terminal repeats and encapsidation sequence When the recombinant vector is a replication-competent adenoviral vector, early genes are in general preserved.
  • the recombinant vector is a replication-defective adenoviral vector, it is preferable that the genome of recombinant adenoviral vector of the invention contains a non-functional El region.
  • a viral gene under consideration may be rendered non-functional by any technique known to the person skilled in the art, in particular by total removal, substitution, partial deletion or the addition of one or more bases to the genes under consideration. Such modifications may be achieved in vi tro on isolated DNA or in si tu, for example using techniques of genetic manipulation or by treatment with mutagenic agents. Other regions may also be modified, in particular the E3 region (WO95/02697) , the E2 region (W094/2938) , the E4 region (W094/28152, W094/12649 and WO95/02697) and the L5 region (WO95/02697) .
  • An adenovirus for use in accordance with the present invention, may contain a deletion or multiple deletions, for example, a deletion in the El and E4 regions. Further, an adenovirus of the invention may contain a deletion in the El region into which a nucleic acid of interest is inserted. It is further contemplated that altered VA-RNA nucleic acid sequences may be used in other vector systems and vaccines which are not derived from an adenovirus . For example, VA-RNA ! of AdHu5 may be added to a non-adenoviral vector in order to increase transgene expression of that particular vector.
  • non-adenoviral vectors into which VA-RNA sequences may be inserted include, but are not limited to, retrovirus vectors ⁇ e . g. , U.S. Patent No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980- 990; Miller (1990) Human Gene Therapy 1:5-14; Scarpa, et al. (1991) Virology 180:849-852; Bums, et al . (1993) Proc . Natl . Acad. Sci . USA 90:8033-8037; Boris-Lawrie and Temin (1993) Curr. Opin. Genet. Develop.
  • AAV vectors which may be readily constructed using techniques well-known in the art (see, e.g., U.S. Patent Nos . 5,173,414 and 5,139,941; WO 92/01070 and WO 93/03769; Lebkowski, et al . (1988) Mol. Cell. Biol. 8:3988-3996; Vincent, et al . (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press) ; Carter (1992) Curr. Opin. Biotechnol. 3:533-539; Muzyczka (1992) Curr. Topics Microbiol. Immunol.
  • VEE Venezuelan Equine Encephalitis
  • the invention relates to the general concept of modifying VARNA nucleic acid sequences, rather that modifying VA-RNA nucleic acid sequences from particular serotypes of virus.
  • the VA-RNA nucleic acid sequences from AdC68 are completely homologous to VA-RNA nucleic acid sequences of human adenovirus serotype 4 (AdHu4) (Kidd, et al . (1995) Virology 207:32-45) and are therefore interchangeable when inserted into other vector systems .
  • AdHu4 human adenovirus serotype 4
  • a specific example of the utility of a vector of the present invention includes an HIV-1 vaccine.
  • an immune response is defined as a mucosal, innate, or systemic immune response characterized by induction of a measurable B cell response ⁇ e . g.
  • the immune response may be modulated by increasing or decreasing B cell or T cell responses via manipulating weak, intermediate and active VA-RNA nucleic acid sequences as described herein.
  • Antigens of particular interest include, but are not limited to, antigenic epitopes or proteins from cancerous cells ⁇ e . g.
  • antigens are derived from enveloped or non- enveloped viruses.
  • antigens are derived from viruses including, but not limited to, those from the family Arenaviridae ⁇ e.g., Lymphocytic choriomeningitis virus), Arterivirus ⁇ e.g., Equine arteritis virus) , Astroviridae (Human astrovirus 1) , Birnaviridae ⁇ e.g., Infectious pancreatic necrosis virus, Infectious bursal disease virus), Bunyaviridae ⁇ e.g., California encephalitis virus Group), Caliciviridae ⁇ e.g., Caliciviruses) , Coronaviridae ⁇ e.g., Human coronaviruses 299E and OC43) , Deltavirus ⁇ e.g., Hepatitis delta virus), Fil
  • Measles virus Rubulavirus such as Mumps virus, Pneumovirus such as Human respiratory syncytial virus
  • Picornaviridae ⁇ e.g., Rhinovirus such as Human rhinovirus 1A, Hepatovirus such Human hepatitis A virus, Human poliovirus, Cardiovirus such as Encephalomyocardi is virus, Aphthovirus such as Foot-and-mouth disease virus O, Coxsackie virus) , Poxviridae ⁇ e.g., Orthopoxvirus such as Variola virus), Reoviridae ⁇ e.g., Rotavirus such as Groups A-F rotaviruses) , Retroviridae (Primate lentivirus group such as human immunodeficiency virus 1 and 2) , Rhabdoviridae
  • an antigen is derived from Streptococcus agalactiae, Legionella pneumophilia,
  • Streptococcus pyogenes Escherichia coli , Neisseria gonorrhosae, Neisseria meningi tidis , Pneumococcus ,
  • Hemophilis influenzae B Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii ,
  • Trypanosoma rangeli Trypanosoma cruzi , Trypanosoma rhodesiensei , Trypanosoma brucei , Schistosoma mansoni , Schistosoma japanicu , Babesia bovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia sagina ta , Echinococcus granulosus, Mesocestoides corti I Mycoplasma arthri tidis, M. hyorhinis, M. orale, M. arginini , Acholeplasma laidlawii , M. salivarium, M. pneumoniae, Candida albicans, Cryptococcus neoformans,
  • Suitable transgenes or nucleic acid sequences encoding antigens are well-known to those of skill in the art and may be identified from the GENBANK or EMBL databases.
  • the nucleic acid sequences may encode a protein, peptide, or epitope of an antigen and may have exogenous or endogenous expression control sequences, such as an origin of replication, a promoter, an enhancer, or necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Construction of such nucleic acid sequences is well-known in the art and is described further in Maniatis et al . , Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) .
  • Preferred expression control sequences may be promoters derived from metallothionine genes, actin genes, immunoglobulin genes, CMV, SV40, adenovirus, bovine papilloma virus, and the like.
  • Recombinant viral vectors of the invention may be prepared using any technique known to one of skill in the art (see, for example, Levreto, et al . (1991) Gene 101:195; EP 185 573; Graham (1984) EMBO J. 3:2917). In particular, they may be prepared via well-established molecular clone methodologies or by homologous recombination between a virus and a plasmid which carries, inter alia, the transgene of interest.
  • the homologous recombination is effected following cotransfection of the said virus and plasmid into an appropriate cell line.
  • the cell line which is employed should preferably be transformable by the said elements, and contain the sequences which are able to complement the part of the genome of the replication- defective virus, preferably in integrated form in order to avoid the risks of recombination.
  • Examples of cell lines which may be used include the human embryonic kidney cell line 293 (Graham, et al . (1977) J. Gen . Virol . 36:59) which contains, in particular, integrated into its genome, the left-hand part of the genome of an Ad5 adenovirus (12%) , or cell lines which are able to complement the El and E4 functions (see, e . g.
  • the present invention also includes pharmaceutical compositions containing one or more recombinant vectors dispersed in a physiologically acceptable medium, which is preferably buffered to physiologically normal pH.
  • the recombinant vectors preferably suspended in a physiologically compatible carrier, may be administered to a human or non-human mammalian patient.
  • Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the vector is directed.
  • one suitable carrier includes saline, which may be formulated with a variety of buffering solutions ⁇ e . g.
  • compositions of the invention may contain, in addition to the recombinant vector and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, chemical stabilizers, or for vaccine use, adjuvants.
  • Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
  • Suitable chemical stabilizers include gelatin and albumin.
  • Suitable exemplary adjuvants include, among others, immune-stimulating complexes (ISCOMS) , LPS analogs including 3-O-deacylated monophosphoryl lipid A (Ribi Immunochem Research, Inc., Hamilton, MT) , mineral oil and water, aluminum hydroxide, Amphigen, Avirdine, L121/squalene, muramyl peptides, and saponins, such as Quil A.
  • ISCOMS immune-stimulating complexes
  • LPS analogs including 3-O-deacylated monophosphoryl lipid A (Ribi Immunochem Research, Inc., Hamilton, MT) , mineral oil and water, aluminum hydrox
  • a recombinant vector disclosed herein may be administered in a pharmaceutically effective amount, that is, an amount of recombinant vector that is effective in a route of administration to transfect the desired cells and provide sufficient levels of expression of the selected transgene of interest to provide a therapeutic or vaccinal immune response, e . g. , some measurable level of protective immunity.
  • a pharmaceutically effective amount that is, an amount of recombinant vector that is effective in a route of administration to transfect the desired cells and provide sufficient levels of expression of the selected transgene of interest to provide a therapeutic or vaccinal immune response, e . g. , some measurable level of protective immunity.
  • Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, intranasal, intramuscular, intratracheal, subcutaneous, intradermal, rectal, oral and other mucosal and parental routes of administration.
  • mucosal routes of administration include those which deliver to mucosal tissues, including, without limitation, inhalation, oral, intranasal, vaginal, and rectal routes. Routes of administration may be combined, if desired, or adjusted depending upon the antigen or the disease. For example, in prophylaxis of rabies, the subcutaneous, intratracheal, intranasal and oral routes are preferred. The route of administration primarily will depend on the nature of the disease being treated or prevented. Doses or effective amounts of the recombinant vector will depend primarily on factors such as the condition, the selected nucleic acid sequence of interest, the age, weight and health of the animal, and may thus vary among animals such as humans .
  • the potency of a recombinant vector of the invention may permit the use significantly lower amounts of recombinant vector to provide an effective amount to induce the desired immunogenic effect (e g. , induction of a predetermined level of CD8+ T cells) .
  • an effective dose of recombinant adenovirus may be in the range of about 10 4 and about 10 14 pfu or between about 10 s to 10 16 viral particles.
  • the doses of adenovirus are from about 10 5 to about 10 10 pfu or about 10 6 to about 10 12 viral particles.
  • pfu plaque-forming unit
  • pfu plaque-forming unit
  • the term pfu corresponds to the infective power of a suspension of virions and is determined by infecting an appropriate cell culture and measuring, generally after 48 hours, the number of plaques of infected cells.
  • the techniques for determining the pfu titer of a viral solution are well documented in the literature. It is contemplated that doses may be readily selected, e . g. , depending upon the selected route of delivery and the selected vector system or serotype.
  • the viral vector may be delivered in an amount which ranges from about 10 ⁇ h to about 100 mL, and more preferably, about 1 mL to about 10 mL, of carrier solution containing concentrations of ranging from about 1 x 10 4 plaque forming units (pfu) to about 1 x 10 11 virus/mL, and about 1 x 10 9 to about 1 x 10 11 pfu/mL virus, based upon an 80 kg adult weight.
  • a preferred dosage is estimated to be about 50 mL saline solution at 2 x 10 10 PFU/mL.
  • the therapeutic levels, or levels of immunity, of the selected nucleic acid sequences of interest may be monitored to determine the need, if any, for boosters.
  • the recombinant vector may be delivered using a prime-boost regimen.
  • a variety of such regimens have been described in the art and may be readily selected.
  • One particularly desirable method is described in WO 00/11140.
  • Recombinant vectors of the present invention may also be used for long-term transgene expression in gene therapy approaches . For example, as it is often the case that a limited number of cells are transduced in vivo, an active VA-RNAi nucleic acid sequence may be introduced into any type of gene delivery vehicle in order to increase transgene expression.
  • Nucleic acid sequences or transgenes of particular interest to be expressed in cells of a subject for treatment of genetic or acquired diseases include those encoding adenosine deaminase, Factor VIII, Factor IX, dystrophin, ⁇ -globin, LDL receptor, CFTR, insulin, erythropoietin, anti-angiogenesis factors, glucocerebrosidase, ⁇ -glucouronidase, ⁇ l-antitrypsin, phenylalanine hydroxylase, tyrosine hydroxylase, ornithine transcarbamylase, arginosuccinate synthetase, UDP- glucuronysyl transferase, apoAl, TNF, soluble TNF receptor, interle
  • Cells types which may be modified for gene therapy purposes include hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, skin epithelium and airway epithelium.
  • genes and methods for gene therapy see, e . g. , Wilson, et al . ((1988) Proc . Natl .
  • Gene therapy applications of particular interest in cancer treatment include overexpression of a cytokine gene ⁇ e . g. , TNF- in tumor infiltrating lymphocytes or ectopic expression of cytokines in tumor cells to induce an anti- tumor immune response at the tumor site) , expression of an enzyme in tumor cells which can convert a non-toxic agent into a toxic agent, expression of tumor suppressor genes ⁇ e . g.
  • Gene therapy applications of particular interest in treatment of viral diseases include expression of trans- dominant negative viral transactivation proteins, such as trans-dominant negative tat and rev mutants for HIV or trans-dominant ICp4 mutants for HSV (see e . g. , Balboni, et al. (1993) J. Med. Virol . 41:289-295; Liem, et al . (1993) Hum. Gene Ther. 4:625-634; Malim, et al .
  • RNA molecules such as anti- sense RNAs and ribozymes
  • gene products that are functional RNA molecules such as anti- sense RNAs and ribozymes, may be expressed in a subject for therapeutic purposes.
  • a ribozyme may be designed which discriminates between a mutated form of a gene and a wild-type gene. Accordingly, a "correct" gene ⁇ e . g. , a wild-type p53 gene) may be introduced into a cell in parallel with introduction of a regulated ribozyme specific for the mutated form of the gene ⁇ e . g.
  • vectors similar to those of the present invention may be constructed which instead of, or in addition to, contain other viral proteins ⁇ e . g.
  • Example 1 Transgene Expression Studies Dendritic cells were isolated from mice injected with AdHu5 vectors expressing a transgene.
  • Example 2 Type I IFN Production Splenocytes transduced using standard methods with AdC68 vectors were found to produce elevated amounts of type I IFN, while splenocytes transduced with AdHu5 vectors produce very little type I IFN. Dendritic cells grown in culture or isolated directly from mice were found to produce large amounts of type I IFN when infected with AdC68 vectors but very little amounts of these cytokines were found in cells infected with AdHu5 vectors .
  • Splenocytes transduced with AdC68 vectors did not produce type I IFN when the AdC68 vector was first inactivated prior to transduction. Inactivated vectors are not transcribed and, as a result, dsRNA is not produced.
  • Example 3 Chemical Inhibition of PKR A chemical inhibitor of PKR, 2-aminopurine, was found to increase transgene expression of AdC68 vectors during transduction of dendritic cells grown in culture. Further, 2-aminopurine dramatically decreased the amount of type I IFN production in splenocytes and dendritic cells when said cells were transduced with AdC68 vectors.
  • Example 4 Adenoviral Vectors with Modified VA-RNA Nucleic Acid Sequences
  • an AdC68 molecular clone was created wherein the native AdC68 VA-RNA nucleic acid sequences were removed and replaced with AdHu5 VA-RNA nucleic acid sequences.
  • This recombinant AdC68 vector containing AdHu5 VA-RNA nucleic acid sequences was further modified to encode an ova-np-gfp transgene for use in measuring expression levels and CD8+ responses, and ultimately protection.
  • AdC68 and AdHu5 vectors were modified to encode the same ova-np-gfp transgene.
  • Example 5 Efficacy of Recombinant Viral Vectors with Modified VA-RNA Nucleic Acid Sequences
  • the efficacy of the resulting AdC68-ova-np-gfp, AdHu5- ova-np-gfp, and AdC68w/AdH5 VARNA-ova-np-gfp vectors is evaluated by vaccinating mice having a transgenic T cell receptor that recognizes the ova peptide. Transgene- specific CD8+ responses are measured by intracellular cytokine staining. These mice are also challenged with influenza and protection is provided by the np gene (which is NP of influenza virus) . Dendritic cells are isolated from these mice and GFP expression is measured.
  • vaccinating mice with AdC68 vectors encoding AdH5 VA-RNA may lead to fewer transgene-specific CD8+ cells compared to AdC68 vectors that encode native VARNA transgene.
  • dendritic cells isolated from mice injected with AdC68 vector encoding AdH5 VA-RNA may express more transgene compared to AdC68 vector encoding native VA- RNA. In vi tro experiments are also conducted to evaluate these vectors. Dendritic cells are grown in culture, infected with the recombinant vectors, and transgene expression is determined by GFP expression and western blot analysis. Type I IFN production is also determined using a biological assay.
  • an AdC68 vector with AdH5 VA-RNA will express more transgene in dendritic cells and not stimulate the production of large amounts of type I IFN.
  • an HIV transgene ⁇ e . g. , gag is cloned into the AdC68 recombinant vector that has AdH5 VA- RNA and virus is rescued. Intracellular cytokine staining is completed after vaccinating mice with said vector. It is contemplated that the AdC68 vector with AdHu5 VA-RNA will stimulate fewer transgene-specific CD8+ cells compared to AdC68 vectors encoding native VA-RNA nucleic acid sequences.
  • a rabies transgene ⁇ e . g. , rabies glycoprotein
  • AdC68 recombinant vector that has AdH5 VA-RNA and virus is rescued.
  • Transgene-specific antibody responses are then determined. It is contemplated that an AdC68 vector with AdHu5 VA-RNA will stimulate a greater transgene-specific antibody response compared to AdC68 vector encoding native VA-RNA nucleic acid sequences. Reciprocal viruses to those described above are created by cloning VA-RNA nucleic acid sequences from AdC68 into an AdHu5 recombinant vector.
  • AdHu5 vectors expressing AdC68 VA-RNA will elicit a much stronger CD8+ response and a lower antibody response compared to AdHu5 vectors expressing native VA-RNA.
  • the AdHu5 hybrid vectors may express less transgene and stimulate the production of large amounts of type I IFN when used to transduce dendritic cells. The mechanism by which these recombinant vectors induce an immune response is evaluated with PKR-/- mice and IFNR1 -/- mice.

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Abstract

La présente invention concerne des vecteurs recombinants présentant des séquences d'acides nucléiques d'ARN viral supplémentaires, de remplacement ou modifiées. L'invention concerne également des méthodes d'utilisation desdits vecteurs pour moduler une réponse immunitaire ou pour traiter des maladies génétiques ou acquises.
PCT/US2005/003903 2004-02-06 2005-02-07 Vecteurs recombinants et methodes pour induire une reponse immunitaire WO2005094415A2 (fr)

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Cited By (11)

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US20160008488A1 (en) * 2007-05-09 2016-01-14 Nature Technology Corporation Vectors And Methods For Genetic Immunization
WO2008153733A2 (fr) * 2007-05-29 2008-12-18 Nature Technology Corporation Vecteurs et méthodes pour immunisation génétique
WO2008153733A3 (fr) * 2007-05-29 2009-05-14 Nature Technology Corp Vecteurs et méthodes pour immunisation génétique
EP2333091A3 (fr) * 2007-05-29 2011-10-05 Nature Technology Corp. Vecteurs et méthodes pour immunisation génétique
AU2008262478B2 (en) * 2007-05-29 2014-03-20 Aldevron, L.L.C. Vectors and methods for genetic immunization
AU2008262478C1 (en) * 2007-05-29 2014-06-19 Aldevron, L.L.C. Vectors and methods for genetic immunization
US9109012B2 (en) 2007-05-29 2015-08-18 Nature Technology Corporation Vectors and method for genetic immunization
US20150344909A1 (en) * 2007-05-29 2015-12-03 Nature Technology Corporation Vectors And Methods For Genetic Immunization
US9737620B2 (en) * 2007-05-29 2017-08-22 Nature Technology Corporation Vectors and methods for genetic immunization
EP3246409A1 (fr) * 2007-05-29 2017-11-22 Nature Technology Corporation Vecteurs dépourvus d'antibioresistance
US9950081B2 (en) 2007-05-29 2018-04-24 Nature Technology Corporation Vectors and methods for genetic immunization

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